PROJECT SUMMARY Microsporidia are widely-dispersed, spore-disseminated foodborne/waterborne pathogens causing diverse afflictions in humans including diarrhea, bronchitis and encephalitis. These opportunistic fungal pathogens, which affect primarily immunocompromised individuals, have emerged as important pathogens of HIV patients. Human-infecting NIAID Category B species from the genus Encephalitozoon can infect by endocytosis, la Candida albicans, but this process is poorly understood. Pathogens that infect by endocytosis greatly benefit from attachment to host cells and many fungal species including yeast encode proteins with cell adhesive properties in their subtelomeres. Subtelomeres also play a crucial role in the development of pathogenic countermeasures against host defenses by encoding multiple copies of virulence factors and antigenic variants, a contingency that provides both a buffer against deleterious mutations and various opportunities for the genesis of new factors from the shuffling of small building blocks via recombination. Unfortunately, our knowledge of what is encoded in the subtelomeric regions of human-infecting Encephalitozoon is lacking but even in the sequenced chromosome cores, about half of the predicted proteins could not be assigned any putative function due to their high level of sequence divergence, such that we have no idea what half of their proteome does. This lack of knowledge greatly limits our understanding of how these pathogens can infect us and, for example, which Encephalitozoon protein(s), if any, can specifically trigger the endocytic uptake from their host is unknown. We have previously shown that the closest relative of Encephalitozoon spp., the microsporidium Ordospora colligata, has acquired a septin from its host that can act as such a trigger, and I hypothesize that the Encephalitozoon genomes encode one or more proteins that can perform this role. Furthermore, despite its importance as regulatory on/off expression switches in eukaryotes and usefulness in assessing the function of the corresponding genetic loci, the microsporidian methylome has never been investigated. Here I propose to identify many of the virulence factors that are left to be discovered in the human-infecting Encephalitozoon spp. by determining their complete sequence from telomere-to-telomere, by improving functional predictions using novel in silico approaches, and by sequencing their methylome. From a practical perspective, knowing the methylation state of microsporidia will not only lead to a better functional understanding of the corresponding loci but also greatly help sequencing-based infection diagnostics by facilitating the selection/development of appropriate DNA enrichment kits.